This competitive renewal application continues to focus on the early stages of retroviral replication. The overarching goal of this application is to uncover critical cell-virus interactions that determine the success or failure of retroviral gene expression in human cells, as modulated by epigenetic silencing. It was established decades ago that the prototype avian sarcoma virus (ASV) DNA provirus is subject to epigenetic silencing in mammalian cells, and this system will continue to be studied as a model. The applicants have discovered that epigenetic silencing of ASV can be mediated by the rapid engagement of an antiviral host adaptor protein, Daxx, which recruits the cellular epigenetic silencing machinery. Furthermore, through development and implementation of an innovative, robust, and comprehensive siRNA-based screen, a number of novel and expected human host cell factors that play a role in this response were identified. These findings have opened up the exciting new areas of investigation to be pursued in this competing renewal. In Aim 1, the molecular mechanisms that govern Daxx function as an antiviral factor will be determined. Results of these studies will provide valuable insight into general intrinsic host defense mechanisms. In Aim 2 mechanistic details for the function of several novel factors identified by siRNA screening will be studied, with a focus on those likely to engage viral DNA sequences. The hypotheses that specific viral DNA binding factors either participate in nucleation of silencing factors or act as a transcriptional barrier, will be tested. Aim 3 will exploit new comprehensive and systematic approaches to distinguish the physical and functional epigenetic features of silent proviruses that are established soon after infection (initiation) and after long-term passage (maintenance). Specific hypotheses concerning the roles of antiviral factors, and the interplay of proviral and cellular chromatin, in silencing will be considered. Epigenetic mechanisms have an essential role in the regulation of gene expression during development, in differentiated adult tissues, and in human disease, including cancer. Consequently, results from the proposed research will be highly relevant to virology, as well as the cellular biology of normal and cancer cells. PUBLIC HEALTH RELEVANCE: Cells employ epigenetic silencing mechanisms to repress expression of retroviral genes, and the proposed research will increase our understanding of how such silencing is initiated and maintained in human cells. The expression of cellular genes is also regulated by epigenetic mechanisms during development, and failure or inappropriate silencing can lead to developmental errors and diseases such as cancer. These studies will help to uncover new strategies by which appropriate silencing may be restored, and disease prevented or treated.